Lung surfactant compositions with dynamic swelling behavior

ABSTRACT

Lung surfactant compositions are provided which can form a swelling phase when dispersed in a medium containing electrolytes. Hereby, a more active spreading of the lung surfactant into the alveoli can be obtained after administration to the lungs. Further provided are a pharmaceutical composition and a pharmaceutical kit comprising a lung surfactant composition as well as to a method for the treatment, prevention and/or diagnosis of respiratory distress syndrome or other pulmonary diseases that are associated with a deficiency of a lung surfactant.

FIELD OF THE INVENTION

The present invention relates to lung surfactant compositions which arecapable of forming a dynamic swelling phase when dispersed in a mediumcontaining electrolytes. The dynamic swelling process can be observed bypolarising microscopy and results in formation of a birefringent networkor tubules at an air/liquid interface. The dynamic swelling processresults in a spreading of the lung surfactant over an increased surfacearea compared to the spreading of the lung surfactant in a non-dynamicswelling phase. The spreading takes place during a specific span of timeafter dispersion of a lung surfactant in e.g. a physiologicalelectrolyte solution. Hereby, a more active spreading of the lungsurfactant into the alveoli can be obtained after administration to thelungs, which in turn opens the possibility to use such a composition asa carrier for therapeutically, prophylactically and/or diagnosticallyactive substances into the lungs or other organs or body areas that arehard to access.

The invention also relates to a pharmaceutical composition and apharmaceutical kit comprising a lung surfactant composition as well asto a method for the treatment, prevention and/or diagnose of respiratorydistress syndrome (IRDS or ARDS) or other pulmonary diseases that areassociated with a deficiency of a lung surfactant.

BACKGROUND OF THE INVENTION

Lung surfactants (LS) are complex and highly surface-active materialscomposed of lipids and proteins that are found in the fluid lining thealveolar surface of the lungs. Their principal property is to reduce thesurface tension in the lungs, which is achieved through the presence ofthe lipids as an organised structure at the air-liquid interface in thealveoli. LS prevents alveolar collapse at low lung volumes and decreasesthe work of breathing during normal and forced respiration (biophysicalfunctions). In addition, it is involved in the protection of the lungsfrom injuries and infections caused by inhaled particles andmicroorganisms (immunological, non-biophysical functions). LS issynthesised and secreted by alveolar type II cells. (For a review, seeRobertson and Taeusch, 1995.)

The constitution of a lung surfactant may vary with various factors suchas species, age, and general health conditions of the subject. Variousnatural and synthetic constituents can substitute for each other in asurfactant. Therefore, even a non-rigorous definition of what the lungsurfactant is and what should be included in a lung surfactant fortherapeutic use is dependent on the situation. Surfactants isolated fromlung lavage of healthy mammals contain about 10% protein (half of whichis surfactant specific), and about 90% lipids, of which about 80% arephospholipids and about 20% are neutral lipids, including about 10%unesterified cholesterol. The phospholipid fraction contains mostly(about 76%) phosphatidylcholine (PC), about two thirds is dipalmitoylphosphatidylcholine (DPPC), and the rest is unsaturated. About 11% ofthe phospholipids are made up of phosphatidylglycerol (PG), about 4%phosphatidylinositol, about 3% phosphatidylethanolamine, about 2%phosphatidylserine, about 1.5% sphingomyelin and about 0.2%lysophosphatidylcholine. Surfactant protein A (SP-A) represents 4% ofsurfactant and SP-B and SP-C and SP-D each make up less than 1%,according to current estimates.

SP-A and SP-D belong to the collectin subgroup of the G-type lectinsuperfamily. SP-A binds dipalmitoyl phosphatidylcholine and SP-D bindsphosphatidylinositol. SP-A also interacts with alveolar type II cells,implicating SP-A in surfactant phospholipid homeostasis. SP-A isrequired for the formation of tubular myelin from secreted lamellar bodymaterial.

Surfactant deficiency remains the most common and serious pulmonaryaffliction of premature infants. Surfactant deficiency is the majorfactor responsible for respiratory distress syndrome of the newborn(IRDS) and for adult respiratory distress syndrome (ARDS). Since the1960′s, the exogenous administration of lung surfactant for thetreatment of these syndromes has been studied.

A pathophysiologic role for surfactant was first appreciated inpremature infants with respiratory distress syndrome (IRDS) and hyalinemembrane disease Use of exogenous lung surfactant and corticosteroidadministration has made a major impact on improving survival andreducing morbidity in this disease with consequent alterations in theclinical and radiographic course.

Initial attempts at improving the treatment of RDS with lung surfactantreplacement during the 1960′s (Chu et al., 1967) failed, largely becauseof a lack of knowledge about lung surfactant compositions anddistributions. Liggins and colleagues (Liggins et al., 1972) were thefirst to utilise corticosteroids for the enhancement of foetal lungmaturation, thereby reducing the risks and complications of RDS afterbirth. It is feasible that combining corticosteroids withthyroid-releasing hormone will enhance prenatal prophylaxis for RDS, andalso inositol can be given as a substrate for lung surfactant productionto infants in the early course of RDS.

A number of approaches for the design and the use of lung surfactantreplacement for RDS have also been tried. The most straightforwardapproach is to replace with human lung surfactant. Human pulmonary lungsurfactant can only be harvested by lavage procedures, though, which maydisrupt its preexisting biophysical and biochemical microorganisation.As seen in a study by Hallman and co-workers, (Hallman et al., 1983),such a preparation was successful in clinical trials, but because of thedifficulties in obtaining large quantities of human lung surfactant, itis not in commercial production.

These limitations make the production of synthetic lung surfactantdesirable A second approach is therefore to learn the functions of thevarious lung surfactant constituents and then construct lung surfactantsthat might be more easily obtained or less expensive than the isolationof the natural products.

Exosurf is a commercially available preparation containing DPPC,hexadecanol and tyloxapol. Hexadecanol and tyloxapol mimic, to somedegree, the functions of surfactant proteins, PG and other lipids innatural lung surfactant. Several groups have added surfactant proteinsto lipids, designing the proteins to mimic structure and function ofnative surfactant proteins.

Furthermore, there are new strategies that add surfactant proteins tolipid mixtures that include formulating proteins using de novo peptidesynthesis or recombinant DNA techniques (Yao et al., 1990)

An ideal therapeutic lung surfactant should share many of the attributesof any ideal therapy. It should be stable, readily available, easy tomake, inexpensive and have an easy route of administration, a half-lifeconsonant with the disease process, and fully understood mechanisms ofaction, metabolism and catabolism. It should have maximum efficacy forthe disease without toxicity, intolerance, immunogenicity or sideeffects. It should mimic the effects of the natural lung surfactant,improve the gas exchange in the lungs, improve lung mechanics, improvefunctional residual capacity, resist inactivation, display optimaldistribution characteristics, and have a known clearance mechanism. Itsuse should completely reverse the primary disease process and repair orallow the body to repair secondary damage from the primary disease.

Available therapeutic lung surfactants are of two types: those that areprepared from mammalian lungs and those made from synthetic compounds.Bovine and porcine surfactants contain SP-8 and SP-C, associated withphospholipids, but SP-A and SP-D are only present in the whole naturalsurfactant. Examples of synthetic lung surfactants that are commerciallyavailable at present are Exosurf and ALEC.

The commercially available lung surfactants are mostly presented asready-mixed liquids, but Exosurf and Alveofact are supplied as alyophilised powder that has to be reconstituted with saline before use.

Surfactant therapy is at present an established part of routine clinicalmanagement of newborn infants with IRDS. An initial dose of about 100mg/kg is usually needed to compensate for the deficiency of alveolarsurfactant (lung surfactant) in these babies, and repeated treatment isrequired in many cases. Recent experimental and clinical data indicatethat large doses of exogenous lung surfactant may be beneficial also inconditions characterised by inactivation of lung surfactant, caused by,for example, aspiration of meconium, infection, or disturbed alveolarpermeability with leakage of plasma proteins into air spaces.

The acute response to lung surfactant therapy depends on the quality ofthe exogenous material (modified natural lung surfactant is generallymore effective than protein-free synthetic surfactants), timing oftreatment in relation to the clinical course (treatment at an earlystate of the disease is better than later treatment and may reduce thesubsequent need for mechanical ventilation) and mode of delivery (rapidinstillation via a tracheal tube leads to a more uniform distributionand is more effective than slow airway infusion). Treatment withaerosolised surfactant improves lung function in animal models ofsurfactant deficiency, but is usually associated with large loss of thenebulised material in the delivery system. Furthermore, data fromexperiments on immature newborn lambs indicate that treatment responsemay depend on the mode of resuscitation at birth, and that manualventilation with just a few large breaths may compromise the effect ofsubsequent surfactant therapy. The widespread clinical use of lungsurfactant has reduced neonatal mortality and lowered costs forintensive care in developed countries.

The most efficient lung surfactants at present are prepared frommammalian lungs. The yield is very low and the therapy is therefore veryexpensive. Therefore there is an urgent need to improve their efficiencyand to standardise their application.

SUMMARY OF THE INVENTION

The present invention provides a lung surfactant composition comprisinga lung surfactant, which—when dispersed as powder or particles in 0.9%w/w sodium chloride in a concentration of 10% w/w at ambienttemperature—is capable of forming, in the course of swelling, abirefringent network or tubules at an air-liquid-solid interface withina time period of from about 0.5 to abut 120 min such as, e.g. from about3 to about 60 min as observed by polarising microscopy.

The birefringent network or tubules are formed during a dynamic swellingprocess that takes place in the span of time from the lung surfactant isdispersed in a medium containing electrolytes and to the steady-state ofswelling is reached (i.e. at equilibrium).

Thus, in another aspect the invention relates to a lung surfactantcomposition, which—when dispersed as a powder or as particles in anelectrolyte solution having an ionic strength of at least about 5 mMsuch as, e.g., at least about 10 mM, at least about 15 mM, at leastabout 20 mM, at least about 25 mM, at least about 50 mM, at least about75 mM, at least about 100 mM or at least about 125 mM or at an ionicstrength corresponding to physiological conditions, and the thusobtained dispersion has a concentration of water of at least about 55%w/w such as, e.g. at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95% or at least about 98% w/w,—issubject to a dynamic swelling process during which a birefringentnetwork or tubules are formed, as observed by polarising microscopy, andthe dynamic swelling process ends when steady-state is reached.

When dispersed in an electrolyte solution a liquid crystalline lamellarphase of the phospholipids is formed. The lipid-protein bilayerstructure has been found to be organising towards an activeestablishment of an equilibrium composition, which is microscopicallyperceptible as a formation of a birefringent complex network. A LScomposition for administration e.g. at a predetermined time point orduring a well-defined time period is provided as well as means fordetermining said optimal time point or time period for a lung surfactantcomposition.

A more active spreading of a lung surfactant into the alveoli can beobtained when applying to the lungs or other parts of the respiratorysystem a lung surfactant that has dynamic swelling behaviour in a mediumcontaining electrolytes. In this manner an improved treatment,prevention or diagnosis can be obtained.

Furthermore, a lung surfactant composition according to the inventionmay be used as a carrier for therapeutically, prophylactically and/ordiagnostically active substances e.g. for pulmonary drug delivery.

The invention also provides a pharmaceutical composition, apharmaceutical kit and method for an improved treatment of respiratorydistress syndrome (RDS) or other respiratory or pulmonary diseases thatmay be associated with deficiency of surfactant.

In a still further aspect the invention provides an in vitro validationmethod for testing individual batches of a lung surfactant compositionwhich has dynamic swelling behaviour when dispersed in an electrolytesolution, the method comprising

a) determining t_(½) for maximum dynamic swelling as described herein,

b) comparing the thus obtained t_(½) with a in vivo—in vitro correlationcurve, obtained as described herein, and

c) evaluating the batch as acceptable or not acceptable.

The invention also relates to an in vitro method for evaluating thetherapeutic, prophylactic and/or diagnostic effect of a lung surfactantcomposition, which has dynamic swelling behaviour when dispersed in anelectrolyte solution, the method comprising determining the half-life ofthe steady-state swelling and comparing the thus obtained half-life within vivo-in vitro correlation curves in order to predict the therapeutic,prophylactic and/or diagnostic effect.

FIGURES

FIG. 1 shows a sample of 10% w/w PLS and 90% w/w Ringer solution viewedin the polarizing microscope 5 min after mixing. The PLS particles underswelling accumulate at the surface towards air.

FIG. 2 show the sample as shown in FIG. 1 15 min after mixing. The“growing” tubules form branches as a treelike structure.

FIG. 3 shows the same sample as shown in FIG. 1 and FIG. 2 30 min aftermixing. The photo above is taken in ordinary light whereas the photobelow is taken in polarised light.

FIG. 4 shows the results from the animal studies described in Example 2herein.

DETAILED DESCRIPTION

The present invention is based upon the surprising finding that anelectrolyte containing medium, such as for example a Ringer solution ora sodium chloride solution, contrary to pure water, induces a highlydynamic swelling behaviour of a dispersion during the early stages ofdispersing a lung surfactant in the medium. During this swelling period,a lipid-protein bilayer structure is organised towards an activeestablishment of an equilibrium conformation. This process involvesspreading at an interface and can be followed in a polarizing microscopeas formation of birefringent networks, which is illustrated in FIGS. 1-3(described in further detail below). Birefringence is due to theoccurrence of different refractive indices in different directions ofthe sample, and it indicates the occurrence of crystalline orliquid-crystalline order within the sample. Polar lipids like those inlung surfactant are known to form liquid-crystalline phases in watersolutions. It is thus the optical character of these phases that can befollowed by their birefringence.

Lung surfactant composition

Thus, the invention relates to a lung surfactant composition comprisinga lung surfactant, which—when dispersed as powder or particles in 0.9%w/w sodium chloride in a concentration of 10% w/w at ambient and/or atbody temperature—is capable of forming, in the course of swelling, abirefringent network or tubules at an air-liquid-solid interface withina time period of from about 0.5 to about 120 min such as, e.g., fromabout 3 to abut 60 min as observed by polarising microscopy. In contrastto a lung surfactant composition according to the present invention,known and marketed lung surfactant compositions do not possess such adynamic swelling behaviour when dispersed in an electrolyte solution(see Example 4 herein).

The electrolyte-containing medium, in which the dynamic swelling occurs,is normally an aqueous medium containing one or more solvents ordiluents. An excellent example of a solvent is water, which is preferredwhen the lung surfactant composition is administered to the lungs, butthere may be situations where a small amount of other solvents such as,e.g., ethanol, isopropanol or polyethylene glycol can be present.

The dynamic changes that are observed are related to the presence ofions such as positive and/or negative ions, respectively. Theelectrolyte containing medium such as, e.g., an electrolyte solution maycomprise at least one of the following cationic species: Na⁺, K⁺, Li⁺,Ca²⁺, Mg²⁺ and/or NH₄ ⁺ and/or at least one of the following anionicspecies: chloride, acetate, carbonate, hydrogen carbonate, dihydrogenphosphate (H₂PO₄ ⁻), monohydrogen phosphate (HPO₄ ²⁻), phosphate (PO₄³⁻), tartrate, citrate, borate, fumarate, or the like. The electrolytemedium has normally such a constitution that it is physiologicallyacceptable, i.e. it does not harm or injury the body, especially not atthe site of administration. In other words, the medium has normally asalt concentration and a pH, which corresponds to physiologicallyacceptable conditions. With respect to pH it means that the pH is in arange of from about 5 to about 8 and the salt concentration correspondsto that of a 0.9% sodium chloride solution or that of a Ringer orRinger-acetate solution. The electrolyte solution may also be a 0.9% w/wsodium chloride solution, Ringer solution or Ringer-acetate solution.

A suitable electrolyte containing medium may also comprise one or moreinorganic or organic salts, which impart ionic strength to thecomposition when dispersed in an aqueous medium such as, e.g., water.Suitable the inorganic salts for use in a lung surfactant compositionaccording to the invention may be selected from the group consisting ofalkaline metal salt such as, e.g., sodium chloride, potassium chloride,lithium chloride and alkaline earth metal salts such as, e.g. calciumchloride, magnesium chloride etc.

Examples of suitable organic salts for use according to the inventionmay be selected from the group consisting of acetates such as, e.g.,sodium acetate, potassium acetate, lithium acetate, citrates, tartrates,fumarates, borates, phosphates, ammonium salt such as e.g. ammoniumchloride etc.

The ionic strength of a suitable medium for use as a dispersion mediumfor a lung surfactant composition in order to obtain a dynamic swellingbehaviour of the lung surfactant composition is also important. It iscontemplated that the ionic strength should be at least about 5 mM suchas, e.g., at least about 10 mM, at least about 15 mM, at least about 20mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, atleast about 100 mM or at least about 125 mM. The ionic strength iscalculated from the following equation:

I=0.5Σc₁z₁ ²

wherein c₁ is the molar concentration of an individual ion in the mediumand z₁ is the loading of the individual ion. Thus 0.9% w/w sodiumchloride solution corresponds to an ionic strength of about 0.0156 M(15.6 mM) and Ringer-acetate solution has an ionic strength of about0.138 M (138 mM).

The lung surfactant composition according to the invention may alsocomprise the ionic species or salts mentioned above. Especially in thosecases where the lung surfactant composition is in the form of a powderor particles it could be appropriate to add a specific amount of theionic species or salts so that the lung surfactant composition onlyshould be dispersed in water in order to obtain the dynamic swellingbehaviour.

The physiological relevance of the effects of the dynamic swellingbehaviour of a lung surfactant composition according to the inventionhas further been tested in animal studies, showing significant clinicaleffects (see Example 2 herein).

To determine whether a lung surfactant has the ability of dynamicswelling and/or to determine the point in time at which the dynamicswelling phase is at its maximum and/or to determine an optimal point intime for administering a lung surfactant composition in accordance tothe present invention, samples containing LS dispersed in aphysiological electrolyte solution are prepared and samples of thatmixture are regularly examined in a polarising microscope. Normally, theconcentration of the lung surfactant should be from about 0.5 to about45% w/w and the concentration of water should exceed about 55% w/w.Often the water content should be more than 80% w/w, such as about 90%w/w. At first, a homogeneous appearance will be obtained and the samplewill be turbid with a viscosity like water. A view of the sample at thisstage in the polarising microscope will resemble FIG. 1. Small particleswith a weak birefringence surrounded by the electrolyte solution can beseen accumulated at the outer boundary of the liquid phase towardswater. The birefringence will then be observed to increase followed by aremarkable increase of contact surface area of the liquid phase towardsair. Tubular formations grow out from the front of the liquid andgradually branch out to form tree-like structures, which successivelybecome birefringent, comparable to those shown in FIGS. 2 and 3. Atapproximately 60 min (range 0.5-120 min such as, e.g., 3-60 min) afteradding the lung surfactant composition to the physiological electrolytesolution, a sample taken will not exhibit the dynamic swelling behaviourdescribed above.

The term “birefringence” used herein means the separation of light, onpassing through a crystal, into two unequally refracted, plane-polarizedrays (of orthogonal polarizations). This effect occurs in crystals orliquid crystals in which the velocity of light is not the same in alldirections; that is, the refractive index is anisotropic.

The term “network or tubules” refer to a remarkable increase of contactsurface area of the liquid phase towards air resulting in tubules withbranches which, when observed by polarising microscopy, becomebirefringent. The liquid surface may form a tree-like structure and thesurface zone develops into a birefringent complex network (see FIG. 3).

It is contemplated that the time for maximum dynamic swelling varieswith the concentration and nature of the components of a lung surfactantcomposition (e.g. mammalian extract or a semisynthetic or fullysynthetic lung surfactant composition; even between batches of the samelung surfactant composition there may be variations), the method for itspreparation and the composition of the dispersion medium employed (ionicstrength, nature of the ionic species, concentration of the ionicspecies, pH etc), it is necessary to determine the dynamic swellingprocess and the point in time or time period for maximum dynamicswelling by a standardised procedure. Furthermore, the particle size ofthe lung surfactant composition is also important. Thus, it iscontemplated that a reduction in particle size will lead to a fasterdynamic swelling, i.e. the time to obtain steady state as well as thetime to obtain maximum dynamic swelling will decrease. This feature maybe used when it is desired to have a specific and well-defined timeperiod for obtaining maximum dynamic swelling.

The lung surfactant used in the present invention are preferably derivedfrom porcine lung, i.e. a porcine lung surfactant (PLS), but as theperson skilled in the art will easily comprehend, they can as welt bederived from other mammalian origin, or even be synthetically produced.In one embodiment of the invention, the PLS is prepared from freshlyslaughtered pigs. The pig lungs are minced and washed in saline solutionand the mixture of proteins and lipids are then filtrated andcentrifuged. Successively, the supernatant is centrifuged and the pelletof crude surfactant extracted as described (Bligh et al., 1959). Theorganic solvent phase is evaporated and neutral lipids removed byacetone. The preparation may finally be freeze-dried so that alyophilised powder is obtained.

In one embodiment of the invention, the lung surfactant composition isobtained from porcine lungs (Leo Pharmaceutical Products, Ballerup,Denmark) but the surfactant can as well be obtained from alveolar cellcultures, or alternatively, be obtained chemically or synthetically e.g.by use of recombinant techniques.

Lung surfactant lipids and/or proteins can be obtained by culturing lungcells and harvesting the secreted lipids and/or proteins by methods wellknown to a person skilled in the art. Cell culture of lung surfactant ise.g. possible by use of the available ATCC cell line A549 (ATCC, 10801University Boulevard, Manassas, Va. 20110-2209, USA), which is derivedfrom a human adenocarcinoma of the lung. One embodiment of thisinvention therefore relates to the use of a lung surfactant derived froma cell line.

The extract described above contains hydrophobic proteins andphospholipids. It may further contain cholesterol, free fatty acids andfatty acid glycerides. In one embodiment of the invention, the lungsurfactant composition may comprise surfactant proteins and lipids thatare selected from a group consisting of phospholipids, DPPC, PG, fattyacids, SP-B and SP-C. In another embodiment of the invention, the lungsurfactant composition may further comprise synthetic phospholipids andat least one of the hydrophobic proteins SP-B or SP-C. Said proteins mayalso be obtained as recombinant proteins.

Both SP-B and SP-C present in the LS extract used in the presentinvention are basic proteins under physiological pH-values, like thosefrom nerve myelin. The lipids are both anionic, which can formelectrostatic complexes with the cationic protein, and zwitterionic(PC).

As mentioned above, a lung surfactant composition according to theinvention comprises phospholipids such as, e.g. saturated andunsaturated phospholipids or mixtures thereof. The concentration ofphospholipids is from about 80 to about 99.5% w/w such as, e.g. fromabout 85 to about 98% w/w or from about 90 to about 98% w/w of thecomposition in dry form. The phospholipids may comprise dipalmitylphosphatidylcholine (DPPC). Furthermore, a lung surfactant compositionaccording to the invention comprises surfactant proteins such as, e.g.,SP-A, SP-B, SP-C and/or SP-D, preferably SP-B and/or SP-C. The totalconcentration of surfactant proteins is from about 0.5 to about 10% w/w,such as, e.g., from about 0.5% to about 7.5% w/w, from about 0.5 toabout 5% w/w, from about 0.5 to about 2.5% w/w or from about 0.5% toabout 2% w/w of the composition in dry form.

Before administration of a lung surfactant composition according to thepresent invention the lung surfactant composition may be dispersed in anaqueous electrolyte medium. This is performed by adding an electrolytemedium, for example Ringer solution, to a predetermined amount of lungsurfactant composition, into a glass test tube. The dispersion is suckedup and ejected by a syringe repeatedly during about 0.5 min in order tofacilitate mixing and interaction towards equilibrium. In a specificembodiment of the invention, Ringer-acetate from Pharmacia & Upjohn(Sweden) is used, which contains 130 mmol Na⁺, 4 mmol K⁺, 2 mmol Ca²⁺, 1mmol Mg²⁺, 30 mmol Ac⁻, and 100 mmol Cl⁻. In another embodiment, saidphysiological electrolyte solution further comprises SP-A.

The microscopic observations of the dynamic swelling processes of a lungsurfactant composition according to the present invention indicate thatboth the presence of ions and the presence of an air/solid/liquidinterface is needed for the remarkable formation of a surface networktextures as seen in FIGS. 2 and 3. This process represents a dynamicallyactive state of a lung surfactant. Without being bound to any theory itis contemplated that extraction in an organic solvent and evaporation,as in the LS extract or preparation used in the present invention, meansthat polar regions are turned inside and hidden by hydrocarbon regions.The PG/SP-B and PG/SP-C ionic complexes are therefore assumed to changetheir conformations drastically in order to form bilayers, when exposedto water. This process takes some time. When ions from e.g., saline orRinger solution are present, they may contribute to the dissociation ofthese complexes. This mechanism also explains why the network is notobserved in lung surfactant samples swollen in distilled water.

Any reorganisation within the bilayer is to be expected to induceincreased dynamics. This is probably the reason behind the elaboratebirefringent network formation following the dispersion of LS powder orparticles into Ringer solution. There seems to be a driving force atexposed interfaces to reduce the surface free energy, and thereorganisation process should favour the formation of low-energyinterfaces towards solid surface, liquid and air. In embodiments of thepresent invention where it is suitable to employ a physiologicalelectrolyte solution, the electrolyte solution is selected from thegroup consisting of saline (physiological sodium chloride) solution,Ringer and/or Ringer-acetate solution.

The unexpected physiological effects described above provide a new andimproved means for the clinical use of lung surfactant compositionspossessing a dynamic swelling behaviour. According to the presentinvention, the lung surfactant composition should thus be administeredinto the lungs together with a physiological electrolyte containingsolution in a time-controlled fashion. Alternatively, the lungsurfactant composition can be administered as a powder or as particlesby means of e.g. a powder inhaler and then, the dynamic swelling processmay occur in situ after application. If necessary, the administrationmay be supplemented by a subsequent administration of a suitable mediumin the form of a neubulised liquid in order to enable a localiseddynamic swelling behaviour of the lung surfactant composition. Use ofpowder inhalators may be especially useful when treating or preventingasthma, bronchitis or related respiratory conditions.

Thus, in other aspects the invention relates to the use of a lungsurfactant composition according to the invention for the preparation ofa medicament for the treatment or prevention of infant respiratorydistress syndrome (IRDS), adult respiratory distress syndrome (ARDS),congenital diaphragmatic hernia, acute lung injury, patients treatedwith Extracorporeal Membrane Oxygenation and/or meconium aspirationpneumonia, or for the treatment or prevention of chronic obstructivelung disease, asthma, acute bronchitis, chronic bronchitis,bronchopulmonary dysplasia, lung infections, persistent pulmonaryhypertension, lung hypoplasia, cancer, cystic fibrosis, alveolarproteinosis and/or congenital SP-B deficiency.

A suitable medicament may be prepared by dispersing the lung surfactantin powder or particulate form in a suitable dispersion medium, and thedispersing may be performed in a sufficient period of time to ensuredynamic swelling and formation of a network or tubules. A sufficientperiod of time is from about such as, e.g., from about 1 to about 100min, from about 2 to about 90 min, from about 2 to about 80 min, fromabout 2 to about 70 min, from about 3 to about 60 min, from about 3 toabout 50 min, from about 3 to about 45 min, from about 5 to about 40min, from about 5 to about 35 min, from about 10 to about 35 min, fromabout 15 to about 35 min or from about 20 to about 35 min.

Pharmaceutical compositions and pharmaceutical kits

The present invention also relates to a pharmaceutical compositioncomprising a lung surfactant composition according to the invention. Thepharmaceutical composition may be in solid (e.g. powder, particles,granules, sachets, tablets, capsules etc.), semi-solid (gels, pastesetc.) or liquid (solutions, dispersions, suspensions, emulsions,mixtures etc) form and adapted for administration via e.g. therespiratory organs. A pharmaceutical composition may thus be in powderor particulate form adapted to be dispersed in an aqueous medium beforeuse.

A pharmaceutical composition in liquid form may be in the form of adispersion comprising the lung surfactant composition and an electrolytesolution such as, e.g. a composition that is adapted to physiologicalconditions e.g. a physiologically acceptable solution.

A pharmaceutical composition according to the invention may furthercomprise another therapeutically, prophylactically and/or diagnosticallyactive substance.

In another aspect, the invention relates to a pharmaceutical kitcomprising a first and a second container, the first containercomprising a lung surfactant composition according to the invention andthe second container comprising a dispersion medium for the lungsurfactant composition, accompanied by instructions for dispersing thelung surfactant composition in the dispersion medium before use.

The lung surfactant composition contained in the kit may be in powder orparticulate form.

A pharmaceutical kit according to the invention may include instructionswith recommendations for the time period during which the lungsurfactant composition should be administered after dispersion in thedispersion medium.

The dispersion medium in a pharmaceutical kit according to the inventionmay be an electrolyte solution such as, e.g. a physiologicallyacceptable electrolyte solution such as, e.g. 0.9% w/w sodium chloridesolution, Ringer solution or Ringer-acetate solution.

Furthermore, a pharmaceutical kit according to the invention may alsocomprise another therapeutically, prophylactically and/or diagnosticallyactive substance.

In a specific embodiment the invention relates to a pharmaceutical kitcomprising a first and a second container, the first container being inthe form of an inhaler or the like comprising a pharmaceuticalcomposition according to the invention, and the second container beingin the form of a nebuliser or the like comprising an appropriate medium,which—when administered after administration of the pharmaceuticalcomposition of the first container—ensures formation of a suitable insitu microenvironment for a dynamic swelling process.

For easy administration in clinical use, the present invention alsoencompasses a three component kit for time controlled administration ofa lung surfactant composition, wherein a physiological electrolytesolution is achieved in the administered composition, containing

a) a first component comprising a lung surfactant composition,

b) a second component comprising a salt and, optionally, a dispersionmedium, and

c) a third component comprising a written instruction containinginformation about the period of time during which the aqueous swellingof lung surfactant composition takes place and how to use said kit.

As described above, the lung surfactant composition may contain varyingmixtures of phospholipids and the proteins SP-B and/or SP-C. In aspecific embodiment of the invention, the lung surfactant composition isdispersed in Ringer solution before administration.

The concentration of a lung surfactant composition in a pharmaceuticalcomposition or in the ready to use pharmaceutical composition preparedfrom a kit is generally within the range of 0.5-300 mg/ml LS, such as atleast 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8mg/ml, 9 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, at least 100mg/ml, at least 125 mg/ml, at least 150 mg/ml, at least 175 mg/ml or atleast 200 mg/ml.

The pharmaceutical compositions and kits may be prepared by methods wellknown by a person skilled in the art.

Therapeutic, prophylactic and/or diagnostic use of a lung surfactantcomposition of the invention

The present invention also relates to the use of a lung surfactantcomposition (which may contain a mixture of lipids and proteins from alung extract, or a semisynthetic or even a fully synthetic lungsurfactant, said mixture being dispersed in a electrolyte solution suchas, e.g. a physiologic solution, for the preparation of a compositionfor administration at a predetermined time point or during apredetermined time period after adding said mixture comprising lipidsand proteins to the electrolyte solution.

The present invention thus relates to the use of a lung surfactantcomposition wherein the lung surfactant is derived from a mammalianextract or from a semisynthetic or fully synthetic method, saidcomposition being dispersed in an electrolyte solution, for thepreparation of a composition for administration suitably at apredetermined point in time or during a predetermined time period, afteradding said composition to said electrolyte solution, wherein said pointof time or time period for administration has been determinedmicroscopically as the half-time of the earliest time point at which theswelling behaviour of the dispersion has reached a steady-state. Byadministration into the alveoli at this time maximal use is made of thedynamic spreading during surfactant molecular reconformation in water,induced by an ionic interaction.

The present invention further relates to a method for determining andstandardising the period of time during which the dynamic swelling of alung surfactant takes place in an electrolyte medium, said methodcomprising adding the lung surfactant composition to the electrolytemedium and observing the dynamic swelling kinetics in a polarisedmicroscope, as described above and in Example 1.

A lung surfactant composition according to the invention may beadministered at any point in time to a patient in need thereof. Thus,the lung surfactant composition may be in the form of a dispersion in anelectrolyte medium (such as, e.g. a physiological electrolyte solution)and it may then be administered at any appropriate time after thedispersion has been made As shown in Example 2 herein it is advantageousto utilize the dynamic swelling behaviour of a lung surfactantcomposition according to the invention in order to achieve an improvedeffect (i.e. the effect is larger or, alternatively, the dose may bereduced in order to achieve the same therapeutic effect).

In those cases where it is suitable to administer the lung surfactantcomposition at its maximum dynamic swelling, the time between mixing thelung surfactant in the form of a powder or particles (e.g. inlyophilised form or in dry form) in physiological electrolyte solutionand the administration of said composition into the lungs for optimaleffect is considered to be in the range of approximately 0.5-120 minsuch as, e.g., from about 1 to about 100 min, from about 2 to about 90min, from about 2 to about 80 min, from about 2 to about 70 min, fromabout 3 to about 60 min, from about 3 to about 50 min, from about 3 toabout 45 min, from about 5 to about 40 min, from about 5 to about 35min, from about 10 to about 35 min, from about 15 to about 35 min orfrom about 20 to about 35 min. The present invention therefore alsorelates to the administration of a lung surfactant composition at apoint in time that is at least 3 minutes and at most 60 minutes afteradding said mixture comprising lung surfactant composition and proteinsto said physiological electrolyte solution, such as at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55,60, 75, 90, 100 or 120 min after adding said mixture, or such as at most3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,45, 50, 55, 60, 75, 90, 100 or 120 min after adding said mixture. Asdescribed above, the optimal point in time for administering saidcomposition will vary according to the quality of the dry powder and thechoice of electrolyte solution that is used, and may even be longer than60 minutes, such as between 60 and 75 minutes, or in certain cases evenlonger.

The time optimum can also vary somewhat between saline and Ringersolution. A good agreement between the biologically determined timeoptimum and the half-value of the time required to reach a steady-statebirefringence front of the formulation as seen in the polarisingmicroscope was found to exist in rat studies, and forms the basis of thepresent invention. From other studies with lung surfactant therapy ofARDS it is known that results from rats can be applied in human therapy.It is therefore concluded that the data derived from the rat studies arerelevant also in humans. No such time effects at administration havebeen observed earlier.

The present invention also offers the possible use of the dynamicswelling behaviour of a lung surfactant composition for determining thequality of an LS composition. Access to an in vitro method to validatethe quality is valuable and may replace time-consuming animal studies.Furthermore, the efficacy of the lung surfactant composition seems to berelated to the performance quality as seen in the dynamic swellingbehaviour.

The optimal concentration for lung administration of a lung surfactantcomposition in saline or Ringer solution is considered to be in therange between from about 4 to about 10% w/v. For use in the presentinvention, a concentration of 5% w/v is considered as suitable withregard to an administration through a tracheal tube and the limitationof administering too large volumes. A preferred embodiment of theinvention thus relates to a composition that contains a lung surfactant,such as at least 4%, at least 5%, at least 6%, at least 7%, at least 8%,at least 9% or at least 10% w/v. A specific embodiment relates to acomposition that contains at least 5% w/v lung surfactant.

One embodiment of the present invention relates to the use of a dynamicprocess of spreading of a lung surfactant composition comprising a lungsurfactant for improved administration of said composition into thealveoli of a subject, characterised by administration of said lungsurfactant composition suitably at a predetermined point in time orduring a predetermined time period after adding the lung surfactant toan electrolyte medium such as, e.g. a physiological electrolytesolution. Said subject can be an animal including a human.

The dynamic swelling behaviour of a lung surfactant compositionaccording to the invention imply that at a clinical use the lungsurfactant composition should be administrated into the lungs togetherwith a physiological electrolyte solution in a time-controlled fashion.Consequently, a method of treating a person in need thereof willcomprise administering a lung surfactant composition dispersed (possiblyreconstituted) in a physiological electrolyte solution, into the alveoliof said person during a predetermined span of time during which thedispersion is displaying an active dynamic spreading.

Such a method as described above can be used for the treatment,prevention or diagnosis infant respiratory distress syndrome (IRDS),adult respiratory distress syndrome (ARDS), congenital diaphragmatichernia, acute lung injury, patients treated with Extracorporeal MembraneOxygenation and/or meconium aspiration pneumonia, or for the treatmentor prevention of chronic obstructive lung disease, asthma, acutebronchitis, chronic bronchitis, bronchopulmonary dysplasia, lunginfections, persistent pulmonary hypertension, lung hypoplasia, cancer,cystic fibrosis, alveolar proteinosis and/or congenital SP-B deficiency.

In an especially preferred embodiment of the present invention,administration is performed via a tracheal tube into the lungs.

Pulmonary drug delivery

A lung surfactant composition, a pharmaceutical composition or apharmaceutical kit according to the present invention may also be usedas a carrier for other therapeutically, prophylactically and/ordiagnostically active substance into body areas that are hard to accessand thus provide an improved transport of substances e.g. over thealveolar wall

Thus, the concept described herein can be used as a pulmonary drugdelivery system for release (e.g. controlled release) oftherapeutically, prophylactically and/or diagnostically activesubstance. Lung surfactants can serve as carriers or as vehicles fordelivery of additional active substances such as, e.g. bronchodilators,anti inflammatory agents, histamine-receptor antagonists, inhalationsteroids including corticosteroids, DNA-ases, immunotherapy includingantibodies, vasodilators, antibiotics, growth factors, drugs enhancingepithelial integrity, factors accelerating lung maturation,mucous-dissolving agents including acetylcysteine, anti-neoplasticdrugs, retinoids, vascular targeting compounds, anti-angiogenicsubstances, peptides, polypeptides, proteins and/or gene-therapyincluding viral vectors and naked DNA. These potential uses of a lungsurfactant composition for pulmonary drug delivery would be applicablein particular in the following diseases: chronic obstructive lungdisease, asthma, bronchopulmonary dysplasia, lung infections, persistentpulmonary hypertension, lung infections, lung hypoplasia,bronchopulmonary dysplasia (retinoids including Vitamin A), respiratorydistress syndrome, cancer, cystic fibrosis, alveolar proteinosis, and/orcongenital SP-B deficiency.

Alternatively, the drug delivery system provided by the presentinvention can of course be as applicable for delivering drugs into asubject in need thereof, even if said subject does not suffer from alung-related disease or a disease related to lung sufficiency. Suchdisease could for illustrative purposes only and not limited to, forexample be either cancer and/or diabetes.

In those cases where a lung surfactant composition according to theinvention is used as a carrier for delivery an active substance to therespiratory organs, the time period in which the dynamic swelling of thelung surfactant composition may deliberately be changed (e.g. by changein particle size, concentration of the lung surfactant composition,concentration of the electrolytes, nature of the ionic species involvedetc.) in order to obtain e.g. modified delivery of the active substanceto the subject. The modified release may be a release that is extendedover a predetermined period of time (it can be from about 4 hours toabout 3-5 days).

Another possible field of use for the present invention is the treatmentof patients after surgery, wherein the composition is applied in orderto prevent or avoid adhesion between tissues in mutual contacts.

The field of pulmonary drug delivery is very active at present. The maindelivery route is the oral delivery route, where many complications havebeen reported, which do not exist in pulmonary delivery, such as e.g.degradation of the drugs by the low pH or any of the enzymes in thegastrointestinal tract. The physiological nature of the surfactant makesit ideal as a vehicle in delivery into the lungs of almost any drug usedsystemically.

Additionally, therapeutic agents based on the present invention maycomprise a pharmaceutical substance encapsulated in surfactantliposomes.

The invention is further illustrated in the following non-limitingexamples.

EXAMPLES Example 1

Compositions of porcine lung surfactant (PLS) and their swellingbehaviour—In vitro observation of a dynamic swelling process

Preparation of a PLS composition

All experiments were performed with a porcine lung surfactant extract(Leo Pharmaceutical Products, Ballerup, Denmark) (PLS) prepared fromfreshly slaughtered pigs. PLS was extracted from minced porcine lungsaccording to a method of Bligh and Dyer (Can. J. Biochem. Physiolol.1959, 37, 911-917). The organic solvent phase was evaporated and neutrallipids were removed by acetone. The preparation obtained was finallyfreeze-dried. The product was obtained as a powder composition that wascomposed of a mixture of saturated and unsaturated phospholipids (90-98%w/w of the powder composition), surfactant proteins SP-B and SP-C(0.5-2.0% w/w) and other lipids (up to 10% w/w). The composition wasused in the following experiments.

Preparation of aqueous samples of PLS

Aqueous samples of porcine lung surfactant (Leo Pharmaceutical Products,Ballerup, Denmark) (PLS) were prepared by adding PLS to water or varyingproportions saline solution or Ringer solution in glass test tubes. Theconcentration of PLS in the electrolyte solution employed was in allexperiments 10% w/w, whereas concentration of the electrolytes in theelectrolyte solution was varied (e.g. 0.9% w/w or a 1.8% w/w sodiumchloride solution. The dispersion was sucked up and ejected by a syringerepeatedly during about 0.5 min to achieve mixing,

In a first experiment Ringer-acetate from Pharmacia & Upjohn was used asa solvent (Na⁺ 130 mmol, W⁺ 4 mmol, Ca²⁺ 2 mmol, Mg²⁺ 1 mmol, Ac⁻ 30mmol, Cl⁻ 100 mmol). A droplet of equilibrated or freshly preparedsamples was transferred to microscope slides for examination eitherduring swelling of the dry PLS powder or after equilibrium had beenreached. A coverslip was put down on the droplet very gently, in orderto avoid air bubble incorporation.

Observations in a microscope were performed at 25° C. and/or at 42° C. ALeitz polarising microscope was used equipped with a Sony CD camera andcolour printer.

In the polarising microscope the swelling behaviour of a samplecontaining 10% (w/w) PLS and 90% Ringer solution was studied. Atdifferent time points samples were taken from the bulk solution and puton microscopy slides with coverslips. After about 5 min a homogenousappearance was obtained, the sample was turbid, and particles with aweak birefringence surrounded by the Ringer solution accumulated at theouter boundary of the liquid phase (FIG. 1). A remarkable increase ofcontact surface area of the liquid phase towards air was seen. Tubularformations were seen at the front of the liquid. The “growing” tubulesformed branches, which successively became birefringent, as shown inFIG. 2 recorded at about 15 minutes after sample mixing. FIG. 3 (aboveand below) shows a surface view after about 30 min in both ordinarylight (above) and polarized light (below). The surface zone haddeveloped into a birefringent complex network. This branching behaviourended after approximately 40-60 min, with some variation from one batchto the other.

When PLS was swollen in physiological saline solution there was asimilar growth of networks at the interface. Two differentconcentrations of sodium chloride were employed, 0.9% w/w and 1.8% w/w,respectively. Irrespective of the sodium concentration employed, anetwork structure like the one seen in FIG. 3 was observed.

This dynamic behaviour with pronounced surface enlargement towardsbirefringent network formation was only observed when PLS was swollen insaline or Ringer solution, not with water. Also, when the water was madeisotonic by the addition of glycerol, PLS swollen in this solution stilllacked the dynamic swelling behaviour shown by the electrolytesolutions.

Further experiments have confirmed that PLS will only form abirefringent network or tubule structure if a certain minimumconcentration of electrolytes is present in the dispersion medium. Inother words, the formation of a birefringent network or tubule structureis dependent on the electrolyte concentration and/or the ion strength ofthe dispersion medium.

As mentioned above some of the experiments were performed at twodifferent temperatures. The dynamic swelling behaviour leading to theformation of a birefringent network or tubule structure was seen at bothtemperatures.

Example 2

Animal experiments—in vivo behaviour of PLS compositions with differentdegrees of swelling

Animal protocol

The protocol was approved by the local Animal Committee of ErasmusUniversity, Rotterdam; care and handling of the animals were inaccordance with the NIH guidelines Sixteen male Sprague-Dawley rats(Harlan, CPH, Zeist, the Netherlands) bodyweight (BW) 240-320 g wereanesthetized with nitrous oxide, oxygen and isoflurane (65/33/2%),tracheotomized and a catheter was inserted into a carotid artery.Anaesthesia was maintained with pentobarbital sodium (Nembutal; AlginBV, Maaassluis, the Netherlands) 60 mg/kg/h i.p. injections;neuromuscular block was produced with pancuronium bromide (Pavulon;Organon Technika, Boxtel, the Netherlands) 2.0 mg/kg/h i.m. Bodytemperature was kept within normal range by mean of a heating pad.

Rats were connected to a ventilator (Servo Ventilator 300,Siemens-Elema, Solna, Sweden) and ventilated with pure oxygen in apressure-controlled mode, frequency 30 bpm, an I/E ratio of 1:2, a peakairway pressure (PIP) of 12 cm H₂O and a positive end-expiratorypressure (PEEP) of 2 cm H₂O. Initially, PIP was increased to 20 cm H₂Ofor 1 min to recruit atelectatic areas. Next, surfactant deficiency wasinduced by repeated whole-lung lavage (BAL) to achieve a PaO₂<85 mm Hg.Just before the first lavage, PIP and PEEP were elevated to 26 and 6 cmH₂O, respectively

Treatment was: exogenous surfactant (35 mg/kg bodyweight dispersed insaline 0.9% w/w 25 mg/ml). The PLS saline mixture was repeatedly drawnin and out of a syringe during 0.5 min. The time of swelling dynamicmaximum (corresponding to t_(½)) for the PLS batch used was establishedto be 20 min. One group of eight rats received surfactant 20 min afterpreparation of the surfactant composition and the other group of eightrats received surfactant 60 min after the preparation of the surfactantcomposition. Surfactant composition (4 ml/kg BW±0.4 ml) was administereddirectly into the endotracheal tube followed by a bolus of air (14ml/kg) (ventilator settings not changed).

Blood samples for measurements of PaO₂ and PaCO₂ were taken from thecarotid artery before BAL and 5 min after the last lavage (directlyfollowed by treatment) and at the following times 5, 15, 30, 60, 90 and120 min after surfactant administration (ABL 505, Radiometer A/S,Copenhagen, Denmark).

After the experiments, the animals were killed with an overdose ofpentobarbital sodium.

Statistical analysis

Statistical analysis was performed using SPSS 10.0 statistical softwarepackage (SPSS Inc. Chicago, Ill.). Inter-group comparisons were analysedwith ANOVA. Intra-group comparisons were analysed with repeated measuresof ANOVA. If ANOVA resulted in p<0.05 a Tukey post-hoc test wasperformed. Statistical significance was accepted at p<0.05.

Results

As discussed above, after 40 min the PLS saline solution samples hadreached a steady state of swelling and at about half-time of thisprocess (i.e. at about 20 min) there is a maximum in the dynamicsinvolved in network formation. Two time windows were therefore chosen(20 and 60 min after mixing with saline) to observe whether the in vitrointerfacial dynamics correlated with in vivo surfactant function.

To optimise the detection of any effect of the swelling behaviour on thesurfactant function in vivo, a low dose of PLS was used. The low dosewas by itself not sufficient to completely restore the induced lunginjury, as shown by the PaO₂ data discussed below.

FIG. 10 shows the PaO₂ levels over time in both groups, which receivedPLS, dispersed either 20 min or 60 min before administration. After PLSadministration PaO₂ improved in both groups, but never reachedpre-lavage during the 120 min study period. There was no difference inthe PaO₂ levels at 5 min after administration and at the end of theexperiment (120 min). However, PaO₂ dropped significantly from 5 to 120min after PLS instillation (p<0.001) in the group in which PLS was mixed60 min before administration. Furthermore, the difference in PaO₂between the two groups at 120 min was also significant (p<0.01).

The conclusion is that surfactant function represented by arterialoxygenation of the maximum swelling condition is superior to the steadystate PLS condition. A possible explanation for the better effect ofsurfactant replacement during the dynamic swelling phase is that thedynamic swelling provides a better distribution of the instilledsurfactant. The tree-like projections seen when dynamic swelling isexamined in in vitro conditions extend over millimeters, i.e., overseveral alveolar diameters.

Very important results of this study are demonstration of a variation ofthe physiological effect at administration in relation to the aqueousmixing time, a time which seems to be directly related to the dynamicsof swelling observed in vitro. This means that lung surfactant extractcompositions should be analysed with regard to the swelling dynamics inorder to determine the maximum in dynamics. In general this time isfound to be about the half time of achievement of steady state. Thispre-determined time, which may vary from one production batch of PLS toanother, could enhance the therapeutic effect after administration.

A few additional rat experiments were done using Ringer solution insteadof saline solution, and they showed the same improvements in oxygenationat administration during the maximum in swelling dynamics compared toadministration when the swelling dynamics had stopped.

When just water with glycerol was used in the PLS composition thetherapeutic effect after administration was dramatically reducedcompared to the electrolyte containing solutions.

Example 3

Dynamic swelling behaviour of PLS as a parameter for quality controlanalysis

The aim of the present study is to develop a suitable in vitro testmethod to determine whether a specific batch of PLS (or any other lungsurfactant possessing a dynamic swelling behaviour as described herein)fulfils predetermined requirements in order to be suitable fortherapeutic, prophylactic and/or diagnostic use. Normally, such a testis performed in animal studies such as those described in Example 2, butsuch tests are expensive and involve the use of test animals. Generally,there is a desire to substitute tests involving test animals with invitro tests, if possible. The results shown in Example 1 and 2 indicatethat an in vivo—in vitro correlation can be established between thetherapeutic effect in vivo and the time for maximum dynamic swelling.

The establishment of such an in vivo—in vitro correlation is typicallyperformed based on results from at least 10 different batches of PLS. Inthe following is described a procedure for determining an in vivo—invitro correlation.

Samples of different batches of PLS prepared as described in Example 1are subjected to the procedure described in Example 1 to investigate thedynamic swelling behaviour of PLS in dispersion. 10% w/W PLS in powderor particulate form is dispersed in 90% w/w of a 0.9% w/w sodiumchloride solution. Samples of the PLS dispersion are observed in apolarising microscope as described in Example 1, and the time at whichbirefringent tubule, branching or network formation occurs in eachsample is noted as is the time when the swelling reaches a steady state.Also, the degree of interfacial length increases, and the strength ofbirefringence may be recorded. Based on the microscopy observations, amaximum dynamic swelling is set for each sample at about half the timerequired to reach steady state swelling Also, the quality of the dynamicswelling behaviour may be classified.

Samples of each PLS dispersion tested microscopically are also tested inthe animal model described in Example 2 to establish a correlationbetween in vitro swelling dynamics and in vivo surfactant function. PLSsamples are instilled in the animals at different points in time afterpreparation of each dispersion, and the effect of PLS instillation isdetermined by measuring the PaO2 level according to the method ofExample 2. The intervals between the time at which the PLS dispersion isprepared and the time at which it is instilled in the animal resultingin a pronounced improvement in PaO₂ levels are noted.

Based on comparisons of said intervals and the time periods observed togive rise to maximum dynamic swelling in vitro, a correlation between invitro behaviour and in vivo effect may be established with a view todefining time periods during which dynamic swelling of PLS dispersionsgive rise to an optimum surfactant effect. Also, the quality of thedynamic swelling behaviour may be taken into account.

Once defined for a representative number of samples, such time periodsmay then be used as standards for validation and/or quality control ofsubsequently produced batches of PLS. The in vivo—in vitro relationship(correlation) can be used in the validation of individual batches of PLSinstead of using time-consuming animal experiments and certain limitscan be set for the point of time at which the maximum dynamic swellingoccurs if the batch is acceptable. The limits are typically set att_(½)±15%, t_(½)±10%, t_(½)±7.5% or t_(½)±5%. This implies that samplesof PLS batches are subjected to the in vitro testing procedure outlinedabove, and batches exhibiting a desired dynamic swelling behaviourcharacterised by formation of birefringent tubules or networks within apredetermined standard period of time are accepted, while batches notexhibiting this behaviour are discarded.

Based on data obtained so far with PLS dispersions, it would appear thatperiods of time within which PLS dispersions may advantageously beadministered are in the range of about 15-30 minutes from dispersing thePLS powder in saline.

Example 4

Swelling behaviour of marketed lung surfactant compositions

Various marketed lung surfactant compositions have been tested withrespect to their swelling behaviour. The following products were tested:Alveofact, Curosurf and Exosurf. Alveofact and Exosurf are in the formof dry powders whereas Curosurf is in the form of a dispersion. Table 1below gives a summary of commercially available lung surfactantpreparations.

TABLE 1 Commercially available surfactant preparations (from D. Gommers.Thesis 1998, University of Rotterdam, “Factors affecting surfactantresponsiveness” Clinical Phospho- doses Preparation Producer CompositionLipids* Proteins (mg/kg) Alec ™ Britannia, Synth. DPPC 100% 0% 100 ( =Pumactant) Redhill, and PG (7:3) England Alveofact* Thomae, Lipidextract  88% 1% 100 ( = SF-RI 1) Biberach, from bovine Germany lunglavage BLES F. Possmayer, Lipid extract  90% 1% 100 Univ. Western, fromcalf lung Ontario, lavage USA Curosurf*^(#) Chiesi, Lipid extract  99%1% 200 Parma, from minced Italy porcine lungs Exosurf* Burroughs- Synth.DPPC,  84% 0% 67.5 Wellcome, hexadecanol, New York, tyloxapol USAInfasurf* Ony Inc., Lipid extract  95% 1% 100 ( = CLSE) New York, fromcalf lung USA lavage Surfacten* Tokyo Tanabo, Lipid extract  84% 1% 100( = surfactant-TA) Tokyo, from minced Japan bovine lungs + synth. DPPCSurvanta ™ Abbott, Lipid extract  84% 1% 100 ( = Beractant) Wiesbaden,from minced Germany bovine lungs + synth. DPPC DPPC,dipalmitoylphosphatidylcholine. PG, phosphatidylglycerol. Synth.,synthetic. *, by weight.

Most of the lung surfactants employed are extracts from calf or bovinelungs. However, Curosurf contains an extract from minced porcine lungswith a relatively high content of phospholipids (99% w/w).

The swelling behaviour was determined by dispersing 50 mg of lungsurfactant in 1000 mg 0.9% w/w sodium chloride as described in Example1, and the dispersions were observed in a polarisation microscope.

All products except Curosurf are in the form of powders. Curosurf is inthe form of a suspension and the swelling behaviour of Curosurf wasdetermined by putting a droplet on a slide with a coverslip.

All products swelled after dispersion in saline. However, none of theproducts investigated in this example exhibited a dynamic swellingbehaviour with formation of a birefringent network, i.e. the products donot swell dynamically as seen with the PLS.

What is claimed is:
 1. An isolated lung surfactant compositioncomprising a lung surfactant, which-when dispersed as powder orparticles in 0.9% w/w sodium chloride in a concentration of 10% w/w atambient temperature-is capable of forming, in the course of swelling, abirefringent network or tubules at an air-liquid-solid interface withina time period of from about 0.5 mm to about 120 minutes as observed bypolarising microscopy.
 2. A method according to claim 1, wherein thelung surfactant composition is administered as a medicament prepared bydispersing the lung surfactant in powder or particulate form in asuitable dispersion medium.
 3. A method according to claim 1, whereinthe lung surfactant composition is administered as a medicament preparedby dispersing the lung surfactant in powder or particulate form in asuitable dispersion medium.
 4. A method according to claim 3, whereinthe mammal is a human.
 5. A method according to claim 3, whereindispersing is performed for a sufficient period of time to ensuredynamic swelling and formation of a birefringent network or tubules. 6.A method according to claim 3, wherein the lung surfactant compositionis administered as a medicament prepared by dispersing the lungsurfactant in powder or particulate form in a suitable dispersionmedium.
 7. A method according to claim 6, wherein the sufficient periodof time is from about 0.5 to about 120 minutes.
 8. A method according toclaim 6, wherein the sufficient period of time is from about 1 to about90 minutes.
 9. A method according to claim 6, wherein the sufficientperiod of time is from about 2 to about 70 minutes.
 10. A methodaccording to claim 6, wherein the sufficient period of time is fromabout 3 to about 45 minutes.
 11. An isolated lung surfactantcomposition, which-when dispersed as a powder or as particles in anelectrolyte solution having an ionic strength of at least about 5 mM orat an ionic strength corresponding to physiological conditions, and thethus obtained dispersion has a concentration of water of at least about55% w/w,- is subject to a dynamic swelling process during which abirefringent network or tubules are formed, as observed by polarisingmicroscopy, and the dynamic swelling process ends when steady-state isreached.
 12. A lung surfactant composition according to claim 11,wherein the electrolyte solution has an ionic strength of at least about10 mM.
 13. A lung surfactant composition according to claim 11, whereinthe dispersion obtained has a concentration of water of at least about60% w/w.
 14. A lung surfactant composition according to claim 11,wherein the lung surfactant-when dispersed in an electrolyte solution-isin the form of a liquid crystalline lamellar phase.
 15. A lungsurfactant composition according to claim 11, wherein the electrolytesolution comprises at least one of the following cationic species: Na+,K+, U+, Ca2−, Mg2+ and/or NH4+.
 16. A lung surfactant compositionaccording to claim 11, wherein the electrolyte solution comprises atleast one of the following anionic species selected from the groupconsisting of: chloride, acetate, carbonate, hydrogen carbonate,dihydrogen phosphate (H₂PO₄), monohydrogen phosphate (HPO₄ ²⁻),phosphate (PO₄ ³⁻), tartrate, citrate, borate and furnarate.
 17. A lungsurfactant composition according to claim 11, wherein the dispersionobtained has a concentration of water of at least about 70% w/w.
 18. Alung surfactant composition according to claim 11, wherein thedispersion obtained has a concentration of water of at least about 80%w/w.
 19. A lung surfactant composition according to claim 11, whereinthe dispersion obtained has a concentration of water of at least about90% w/w.
 20. A lung surfactant composition according to claim 11,wherein the electrolyte solution is a sodium chloride solution.
 21. Alung surfactant composition according to claim 20, wherein theelectrolyte solution is a sodium chloride solution selected from thegroup consisting of: a 0.9% w/w sodium chloride solution, Ringersolution and Ringer-acetate solution.
 22. A lung surfactant compositionaccording to claim 1 or 11 comprising dipalmitylphosphatidylcholine(DPPC).
 23. A lung surfactant composition according to claim 1 or 11comprising at the most up to 10% w/w of other lipids than phospholipids.24. A lung surfactant composition according to claim 1 or 11, whereinthe lung surfactant comprises synthetic components.
 25. A lungsurfactant composition according to claim 1 or 11, wherein the lungsurfactant is obtained from mammalian alveolar cell cultures.
 26. A lungsurfactant composition according to claim 1 or 11 further comprisinganother therapeutically, prophylactically and/or diagnostically activesubstance.
 27. A method for preventing adhesion between tissues inmutual contact comprising application of a lung surfactant compositionaccording to claim 1 or
 11. 28. A lung surfactant composition accordingto claim 1 or 11, wherein the lung surfactant is obtained from amammalian lung.
 29. A lung surfactant composition according to claim 28,wherein the lung surfactant is extracted from the mammalian lung.
 30. Alung surfactant composition according to claim 28, wherein the mammalianlung is cattle, bovine, porcine, monkey or human lung.
 31. A method forthe preparation of a pharmaceutical composition, the preparationcomprising dispersing a lung surfactant composition according to claim 1or 11 until a birefringent network or tubules are formed at anair-liquid-solid interface as observed by polarisation microscopy.
 32. Amethod according to claim 31, wherein dynamic swelling of the lungsurfactant occurs within a time period of from about 0.5 to about 120minutes.
 33. A method according to claim 31 wherein the composition isdried.
 34. A lung surfactant composition according to claim 1 or 11,wherein the lung surfactant comprises phospholipids.
 35. A lungsurfactant composition according to claim 34, wherein the phospholipidsare present in the form of a mixture of saturated and unsaturatedphospholipids.
 36. A lung surfactant composition according to claim 34,wherein the concentration of phospholipids is from about 80 to about99.5% w/w of the composition in dry form.
 37. A lung surfactantcomposition according to claim 34, wherein the concentration ofphospholipids is from about 85 to about 98% w/w of the composition indry form.
 38. A lung surfactant composition according to claim 34,wherein the concentration of phospholipids is from about 90 to about 98%w/w of the composition in dry form.
 39. A lung surfactant compositionaccording to claim 1 or 11 comprising surfactant proteins.
 40. A lungsurfactant composition according to claim 39, wherein the surfactantproteins are SP-B and/or SP-C.
 41. A lung surfactant compositionaccording to claim 39, wherein the total concentration of surfactantproteins is from about 0.5 to about 10% w/w of the composition in dryform.
 42. A lung surfactant composition according to claim 39, whereinthe surfactant protein is a recombinant protein.
 43. A lung surfactantcomposition according to claim 39 comprising surfactant proteinsselected from the group consisting of SP-A, SP-B, SP-C and combinationsthereof.
 44. A lung surfactant composition according to claim 39,wherein the total concentration of surfactant proteins is from about 0.5to about 5% w/w of the composition in dry form.
 45. A method for thetreatment and/or prevention of a lung disease or condition in a mammal,the method comprising administering to the mammal in need thereof aneffective amount of a lung surfactant composition according to claim 1or
 11. 46. A method according to claim 45, wherein the administrationtakes place during a dynamic swelling phase of the lung surfactantcomposition.
 47. A method according to claim 45, wherein the lungdisease or condition is selected from the group consisting of infantrespiratory distress syndrome (IRDS), adult respiratory distresssyndrome (ARDS), congenital diaphragmatic hernia, acute lung injury,patients treated with Extracorporeal Membrane Oxygenation and meconiumaspiration pneumonia.
 48. A method according to claim 45, wherein thelung disease or condition is selected from the group consisting ofchronic obstructive lung disease, asthma, acute bronchitis, chronicbronchitis, bronchopulmonary dysplasia, lung infections, persistentpulmonary hypertension, lung hypoplasia, cancer, cystic fibrosis,alveolar proteinosis and congenital SP-B deficiency.
 49. A pulmonarydrug delivery system comprising a lung surfactant composition accordingto claim 1 or
 11. 50. A pulmonary drug delivery system according toclaim 49, wherein the system further comprises another therapeutically,prophylactically and/or diagnostically active substance.
 51. A pulmonarydrug delivery system according to claim 50, wherein the therapeutically,prophylactically and/or diagnostically active substance comprisespeptides, polypeptides or proteins.
 52. A lung surfactant compositionaccording to claim 1 or 11 further comprising one or more inorganic ororganic salts, which impart ionic strength to the composition whendispersed in an aqueous medium.
 53. A lung surfactant compositionaccording to claim 52, wherein the aqueous medium is water.
 54. A lungsurfactant composition according to claim 52, wherein the one or moreinorganic salts comprises an alkaline earth metal salt.
 55. A lungsurfactant composition according to claim 54, wherein the alkaline earthmetal salt is selected from the group consisting of sodium chloride,potassium chloride, lithium chloride and alkaline earth metal salts. 56.A lung surfactant composition according to claim 52, wherein the one ormore organic salts comprises an acetate.
 57. A lung surfactantcomposition according to claim 56, wherein the acetate is selected fromthe group consisting of sodium acetate, potassium acetate, lithiumacetate, citrates, tartrate, fumarate, borate, and phosphate.
 58. A lungsurfactant composition according to claim 56, wherein the ammonium saltcomprises ammonium chloride.
 59. A pharmaceutical composition comprisinga lung surfactant composition according to claim 1 or
 11. 60. Apharmaceutical composition according to claim 59 in powder orparticulate form adapted to be dispersed in an aqueous medium.
 61. Apharmaceutical composition according to claim 59, wherein thecomposition is adapted to physiological conditions.
 62. A pharmaceuticalcomposition according to claim 59 further comprising anothertherapeutically, prophylactically and/or diagnostically activesubstance.
 63. A pharmaceutical kit comprising a first and a secondcontainer, the first container being in the form of an inhaler or thelike comprising a pharmaceutical composition according to claim 8, andthe second container being in the form of a nebuliser comprising anappropriate medium, which—when administered after administration of thepharmaceutical composition of the first container—ensures formation of asuitable in situ microenvironment for a dynamic swelling process.
 64. Apharmaceutical composition according to claim in the form of a powder orparticles adapted to be administered from an inhaler.
 65. Apharmaceutical composition according to claim 64, wherein the meanparticle size and/or the electrostatic properties of the powder orparticles have been adjusted to conditions required in order to reachspecific sites in the respiratory organs after administration via aninhaler.
 66. A pharmaceutical composition according to claim 59 inliquid form.
 67. A pharmaceutical composition according to claim 66,wherein the liquid is in the form of a dispersion comprising the lungsurfactant composition and an electrolyte solution.
 68. A pharmaceuticalcomposition according to claim 67, wherein the electrolyte solution is aphysiologically acceptable solution.
 69. A pharmaceutical kit comprisinga first and a second container, the first container comprising a lungsurfactant composition according to claim 1 or 11 and the secondcontainer comprising a dispersion medium for the lung surfactantcomposition, accompanied by instructions for dispersing the lungsurfactant composition in the dispersion medium.
 70. A pharmaceuticalkit according to claim 69, wherein the lung surfactant composition is inpowder or particulate form.
 71. A pharmaceutical kit according to claim69, wherein the instructions include recommendations for the time periodduring which the lung surfactant composition should be administeredafter dispersion in the dispersion medium.
 72. A pharmaceutical kitaccording to claim 69 further comprising another therapeutically,prophylactically and/or diagnostically active substance.
 73. Apharmaceutical kit according to claim 69, wherein the dispersion mediumis an electrolyte solution.
 74. A pharmaceutical kit according to claim73, wherein the electrolyte solution is a physiologically acceptableelectrolyte solution selected from the group consisting of 0.9% w/wsodium chloride solution, Ringer solution and Ringer-acetate solution.